I'm performing qPCR on bovine whole blood samples collected 9/2021-11/2021 and stored/thawed a handful of times before initial PCR testing for anaplasmosis locally and then for BLV via PCR at Michigan State before being stored again and sent back to me at my lab for some experimental testing for coxiella burnetii on beef cattle. Upon receiving the samples on dry ice, I subsequently stored them at -80 C before I took them out for a few hours to thaw before sample preparation and extraction using Magmax Express -96 Deep Well Magnetic Particle Processor was performed. Our lab has typically used 18s as the endogenous control for multiple different tests, but I suppose I'm unfamiliar if the specimen type is having an effect on the Ct values? One of my NA plates had several wells with no Ct value for 18s, so I did a 10:1 dilution and a Ct value for 18s did eventually show, but it was still higher than I'd like it to be (Ct ~26). There is also a decent amount of variation between the Ct values for 18s samples being tested on the same plate. I think I either inherited some samples that have some agent causing PCR inhibition, or the whole blood samples are no longer viable due to several rounds of thawing/freezing and a couple of trips across the country for other labs to carry out testing. I'm following our institutional SOP that is actually designed for coxiella burnetii on small ruminants, but the thermo fisher kit I'm using has a similar protocol including specifics on how to prep and test whole blood, however, my lab director insists that the thermo fisher MagMAX™ -96 Viral RNA Isolation Kit can be used with whole blood samples, even though there are other kits specifically made for specimens with more cells that what we use the viral kit for, which is for testing ruminant fetal tissues.
Any help would be greatly appreciated!!!
This kit is designed for isolation of viral RNA and DNA from cell-free, or mostly cell-free samples. Cell culture medium, swab samples, and biological fluids such as serum, plasma, urine, meconium, and nasal fluids can be used with the kit.
• The MagMAX™-96 Viral RNA Isolation Kit procedure accommodates up to 50-µL sample input, which is sufficient for most applications. Sample volumes up to 300 µL can be processed to increase the detection sensitivity of low titer samples using the MagMAX™ Pathogen RNA/DNA Kit (Cat. no. 4462359).
Sir, There are several reasons that could explain why the Ct values for your endogenous control (18s) are high and/or variable for the qPCR you're performing on whole blood samples.
One possible explanation is that the levels of 18s RNA in whole blood samples can vary depending on the age, sex, and health status of the donor. Additionally, the presence of inhibitors in whole blood samples can affect the efficiency of the reverse transcription reaction and the subsequent qPCR amplification.
Other factors that can contribute to high or variable Ct values include inadequate sample preparation, poor RNA quality, and suboptimal primer and probe design.
To address this issue, you may want to consider optimizing your sample preparation protocol, using high-quality RNA isolation methods and optimizing your qPCR assay conditions. You may also want to consider using an alternative endogenous control gene that is more stably expressed in whole blood samples.
Thank you
Khalid Khan thanks for providing several possibilties concerning my current issues with my endogenous control. As far as sample preparation goes, it is possible that the use of the Magmax Express -96 Deep Well Magnetic Particle Processor is failing to extract adequate NA. Early on when I was getting poor Ct values for 18s, I selected a few samples (~13 whole blood specimens from my initial batch) and performed manual extraction using a QIAamp Viral RNA Mini Kit (via a Spin Protocol) with QIAamp Mini Columns to extract NA. The Ct values for 18s were lower than what initially resulted from first using the 96 deep well Magnetic Particle Processor, however this would be extremely time consuming to manually extract all 700 samples. During my second sample extraction with the MagMax 96 MPP, I suspected PCR inhibition due to poor 18s Ct values, so I performed a 10:1 dilution on the NA from the same 90 samplesthat I extracted during the second occasion with the MagMax 96 MPP and found that my 18s Ct values were once again lowered after dilution (from no Ct value to a Ct of ~19).
While my primer/probe design has been excellent for the coxiella burnetii positive control (Ct of 22) using FAM and producing an excellent curve.
For 18s, I've used both primer/probe solutions prepared by fellow lab colleagues and also produced my own mixes, but results have been consistent regardless of the primer/probe mix I've used.
What other/additional endogenous controls would you suggest using instead of 18s for testing for coxeilla burnetii (q fever) bacteria?
I have been using 50 ul of blood specimens when preparing the deep well plates for extraction, in addition to adding 130 ul of lysis binding/solution and 20 ul of bead mix (= 10 ul lysis enhancer + 10 ul NA binding beads). Plus 4 plates of 150 ul wash solutions and 1 plate with 60 ul of elution buffer. These volumes are similar to other protocols but have been modified by our lab director to be our institutional standard operating protocols for testing for coxiella burnetii with fetal tissue samples. Other protocols I've read have called for more volume of all solutions I mentioend previously, especially using at least 100 ul of whole blood. I have thawed with efficiency and also inverted samples 8-9 times and used mini vortex to pulse each tube prior to extracting the 50 ul of specimen, but still continuing to see issues. Any additional help or comments would be appreciated!!
Thanks,
MW
Michael Welle
It's great that you've identified possible issues with your sample preparation and have tried alternative extraction methods to improve your 18s Ct values. If you're still experiencing high and/or variable Ct values for 18s, you may want to consider using an alternative endogenous control gene that is more stably expressed in whole blood samples. Some commonly used endogenous control genes for whole blood samples include GAPDH, ACTB, B2M, and HPRT1. You may want to perform a literature search to see which endogenous control gene(s) have been used in previous studies on Coxiella burnetii. Additionally, it's important to ensure that your qPCR assay conditions are optimized for the endogenous control gene you choose to use. This includes validating the primer and probe design and ensuring that the amplification efficiency is similar to that of your target gene.
Thank you
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